The genome sequence of the jumping weevil, Orchestes rusci (Herbst, 1795)

We present a genome assembly from an individual female Orchestes rusci (the jumping weevil; Arthropoda; Insecta; Coleoptera; Curculionidae). The genome sequence spans 624.00 megabases. Most of the assembly is scaffolded into 12 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 21.73 kilobases in length.


Background
The jumping weevil, or flea weevil, Orchestes rusci (J.F.W. Herbst, 1795) is one of nine species in the genus which were previously included in Rhynchaenus Clairville.O. rusci, along with two other UK species has been assigned to the subgenus Alyctus Thomson, CG (Duff, 2016).In common with most other weevils of the tribe Rhamphini, it possesses strongly developed hind femora adapted for jumping.It can be separated from the other eight Orchestes species by the absence of sharp ventral tubercles on the hind femur.
Ranging from 2.2 to 2.5 mm in length and roughly oval in shape, O. rusci has a black body with yellowish antennae and tarsi.The upper side is sparsely covered in a white pubescence, slightly thicker along the base of the elytral suture and also across the elytrae where they form two irregular but distinct white bands (Figure 1).
Orchestes rusci occurs in mixed deciduous woodland and the drier parts of fens, bogs and heaths where the host plants occur.It is found on birch, probably on both Betula pendula Roth.and B pubescens Ehrh.and on their hybrid, in the UK.The adults overwinter and are active from May to September, peaking in June and July.The species is thought to be univoltine in northern areas (Duff, 2016;Morris, 2012;UK Beetles, 2024).Known from most of the Palearctic region from the Pyrenees north to the UK and Fennoscandia and from Ireland east to Russia and Japan (GBIF Secretariat, 2024).
The females chew into the apical margins of leaves and usually lay a single egg in early to mid-summer.Starting at the tip, the larvae mine the edge of the leaf until about the middle when they turn towards the centre producing a mine that widens along its length.The mine terminates in an almost circular blotch against the midrib in which the larva pupates, having stitched the upper and lower epidermis together.This disc is generally excised and falls to the ground.The new adults emerge from July onwards, overlapping with the previous generation but not reproducing until the following year (UK Beetles, 2024).

Genome sequence report
The genome of an adult female Orchestes rusci was sequenced using Pacific Biosciences single-molecule HiFi long reads, generating a total of 22.47 Gb (gigabases) from 2.67 million reads, providing approximately 34-fold coverage.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data, which produced 114.65 Gbp from 759.25 million reads, yielding an approximate coverage of 184-fold.Specimen and sequencing information is summarised in Table 1.
Manual assembly curation corrected 64 missing joins or misjoins and 11 haplotypic duplications, reducing the assembly length by 0.43% and the scaffold number by 15.38%, and increasing the scaffold N50 by 56.44%.The final assembly has a total length of 624.00 Mb in 120 sequence scaffolds with a scaffold N50 of 81.8 Mb (Table 2).The total count of gaps in the scaffolds is 812.The snail plot in Figure 2 provides a summary of the assembly statistics, while Figure 3 shows the distribution of assembly scaffolds based on base coverage across chromosomes.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.42%) of the assembly sequence was assigned to 12 chromosomal-level scaffolds, representing 11 autosomes and the X sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 3).Chromosome X was assigned based on synteny to Philonthus cognatus (GCA_932526585.2) (Crowley et al., 2023b) and Cetonia aurata (GCA_949128085.1)(Grayson et al., 2023).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Sample acquisition
An adult female Orchestes rusci (specimen ID NHMUK014451660, ToLID icOrcRusc1) was collected from   HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol (Oatley et al., 2023).The DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 31 (Bates et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation (Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.
The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences Sequel IIe instrument.Hi-C data were also generated from whole organism tissue of icOrcRusc1 using the Arima-HiC v2 kit.The Hi-C sequencing was performed using paired-end sequencing with a read length of 150 bp on the Illumina NovaSeq 6000 instrument.
The mitochondrial genome was assembled using MitoHiFi (Uliano- Silva et al., 2023), which runs MitoFinder (Allio et al., 2020) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Assembly curation
The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants (ASCC) pipeline (article in preparation).Flat files and maps used in curation were generated in TreeVal (Pointon et al., 2023).Manual curation was primarily conducted using PretextView (Harry, 2022), with additional insights provided by JBrowse2 (Diesh et al., 2023) and HiGlass (Kerpedjiev et al., 2018).Scaffolds were visually inspected and corrected as described by Howe et al. (2021).Any identified contamination, missed joins, and misjoins were corrected, and duplicate sequences were tagged and removed. he entire process is documented at https://gitlab.com/wtsi-grit/rapid-curation (article in preparation).

Evaluation of the final assembly
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020).This work was done using Nextflow (Di Tommaso et al.The genome assembly and evaluation pipelines were developed using the nf-core tooling (Ewels et al., 2020), use MultiQC (Ewels et al., 2016), and make extensive use of the Conda package manager, the Bioconda initiative (Grüning et al., 2018), the Biocontainers infrastructure (da Veiga Leprevost et al., 2017), and the Docker (Merkel, 2014) and Singularity (Kurtzer et al., 2017) containerisation solutions.
Table 4 contains a list of relevant software tool versions and sources.Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material The manuscript reports on a reference genome for Orchestes rusci, the jumping weevil.This paper contributes a Curculionidae genome to the database-a group that is very specious, with few representatives sequenced.The authors do a good job listing out their methods and results.The only part that was a little difficult to follow was the background section on which genus the jumping weevil is classified.
Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: genomics, transcriptomics, Curculioninae I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 1 .
Figure 1.Photograph of Orchestes rusci (not the specimen used for genome sequencing) by Nikolai Vladimirov.

Figure 2 .
Figure 2. Genome assembly of Orchestes rusci, icOrcRusc1.1:metrics.The BlobToolKit snail plot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 624,046,445 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (118,147,993 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (81,830,918 and 30,363,857 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the endopterygota_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Orchestes%20rusci/dataset/icOrcRusc1_1/snail.

Figure 3 .
Figure 3. Genome assembly of Orchestes rusci, icOrcRusc1.1:Distribution plot of base coverage in ERR11512311 against position for sequences in assembly icOrcRusc1_1.Windows of 100 kb are coloured by phylum.The assembly has been filtered to exclude sequences with length < 2,550,000.An interactive version of this figure may be viewed here.

Figure 4 .
Figure 4. Genome assembly of Orchestes rusci icOrcRusc1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Orchestes%20rusci/dataset/icOrcRusc1_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Orchestes rusci icOrcRusc1.1:Hi-C contact map of the icOrcRusc1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=TsqTbYaDSSmpj65xkMw_3w.

Table 2 . Genome assembly data for Orchestes rusci, icOrcRusc1.1. Genome assembly
(Beasley et al., 2023)ication based on morphology, the species taxonomy was verified by DNA barcoding soon after collection, according to the framework developed byTwyford  et al. (2024).A small sample was dissected from the specimen and stored in ethanol.The tissue was lysed, and the COI marker region was amplified by PCR.Amplicons were sequenced and compared to the BOLD database, confirming the species identification(Crowley et al., 2023a).The standard operating procedures for the Darwin Tree of Life barcoding have been deposited on protocols.io(Beasleyetal., 2023).The remaining parts of the specimen were shipped on dry ice to the Wellcome Sanger Institute (WSI).A DNA barcode was also generated from the PacBio sequencing data at a later stage for sample tracking through the genome production pipeline at the WSI(Twyford et al., 2024).

Table 3 . Chromosomal pseudomolecules in the genome assembly of Orchestes rusci, icOrcRusc1. INSDC accession Name Length (Mb) GC%
Wellcome Sanger Institute -Legal and GovernanceThe materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the '

Darwin Tree of Life Project Sampling Code of Practice', which
can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.

Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.
Point 1: "... usually lay a single egg in early to mid-summer."I believe laying a single egg means per leaf, which is unclear in the text, please check and correct if necessary.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.